Increased translational activity during T cell activation requires increased gene expression of several key translation initiation factors. One factor being studied is eIF2-alpha, reported to have a natural antisense mechanism of regulation. A 1.7 kb region from the promoter for eIF2-alpha was cloned in both the sense and antisense orientation into a luciferase reporter gene construct. Extremely strong promoter activity was measured for the promoter in the antisense direction, compared with very weak promoter activity of the sense direction. Deletion mutations of both promoter orientations have identified potential regulatory regions for both constructs. Footprint analysis, EMSA assays and site-directed mutagenesis are being conducted to characterize these potential regulatory regions. An AP1 element in eIF2-alpha promoter was determined to be functional in regulating eIF2- alpha gene expression by cloning a region of the eIF2-alpha promoter containing that site into a luciferase reporter vector. This construct was transfected into HL-60 cells and found to be induced with PMA. By methylation interference assay, critical G nucleotides were identified and mutants constructed based upon the data that had reduced response in transient transfection assays in HL-60 cells. X-linking studies indicate the presence of a larger number of proteins binding to the larger 67 nt probe when compared with the 27 nt AP1 probe. The larger 67 nt region contains an ETS site, a partial NFkB site, and a consensus TIN1 motif. Some if not all of these may play an important role in determining the higher ordered structure of the alpha-Pal promoter. The identity of these proteins is currently being investigated.